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Multiple interactions between murine cytomegalovirus and mouse lymphoid cells in vitro Loh, Lambert C. 1979

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MULTIPLE INTERACTIONS BETWEEN MURINE CYTOMEGALOVIRUS B.Sc. (Honors in Chemistry) McGill University (Montreal) 1967 Ph.D. (Physical Chemistry) University of California (Berkeley) 1974 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In THE FACULTY OF GRADUATE STUDIES Department of Microbiology We accept this thesis as conforming to the required standard AND MOUSE LYMPHOID CELLS IN VITRO by LAMBERT C. /LOH THE UNIVERSITY OF BRITISH COLUMBIA September 1978 0 Lambert Ching-Hon jfLoh, 1978 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s m a y b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . r> 4. 4- „ x Microbiology D e p a r t m e n t o f _ T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2 0 7 5 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V 6 T 1W5 i i ABSTRACT The purpose of the present project was to study the interactions between murine cytomegalovirus (MCMV) and mouse lymphoid cel l s . A l l experiments were conducted with murine spleen cells infected with MCMV in vitro. Most experiments were done with spleen cells from SWR/J mice though random bred Swiss white mice and C3H/HeJ mice were used occasionally. It was shown that in vitro infection resulted in the formation of infectious centers in only a small percentage of the spleen c e l l population even at high multiplicities of infection,when a proportionately higher number of virus particles were being taken up by the cell s . Virus could be rescued from some of these infected cells by co-cultivation with susceptible mouse embryo fibroblasts, and the emerging infectious virus particles were detectable 40-50 hours after the start of co-cultivation. It appeared that allogeneic stimulation was not essential to virus rescue in our in vitro system, for both syngeneic and allogeneic mouse embryo fibroblasts were equally efficient in effecting virus reactivation from infected spleen c e l l s . A fraction of the infected spleen cells was capable of supporting spontaneous MCMV replication. Such replication was not affected by incubation with the mitogens Con A or LPS prior to or after infection with the virus. However, the presence of an increased number of activated T cells due to Con A stimulation possibly inhibited virus replication to a certain extent. Virus replication was i i i also reduced in the absence of some heat-labile factor in the fetal calf serum. Cell separation techniques such as nylon wool column adherence, plastic adherence, anti-serum treatment and y-xay irradiation, showed that macrophage-like cells were probably involved in harboring MCMV in a latent state although spontaneous replication did take place to a limited extent in such cel l s . Another c e l l fraction, with B-cell-like properties, was capable of supporting spontaneous MCMV replication. Another effect of in vitro MCMV infection on spleen cells was their suppressive effect on the mitogenic responses of such cel l s . Preliminary evidence suggested that the defective con A response might be mediated by macrophages exposed to MCMV. Moreover, the immunosuppressive effect could only be observed after exposure to infectious virus particles, and the degree of suppression of mitogen responses could be increased by using higher multiplicities of infection. In certain cases, a slight stimulatory effect on the spleen cells was observed about 30 hours after infection. In summary, the spleen c e l l population contained c e l l fractions that were capable of harboring MCMV in a latent state and supporting spontaneous v i r a l replication. In addition, the mitogenic responses of infected cells were impaired. iv TABLE OF CONTENTS Page CHAPTER I: INTRODUCTION 1 CHAPTER II: MATERIALS AND METHODS 5 A. Materials . 5 1. Reagents 5 2. Solutions and Buffers 6 3. Growth Media 7 a. Mouse embryo monolayers 7 b. Spleen cells 7 B. Methods . 7 1. Viruses 7 a. Growth of MCMV 8 b. Labelling of MCMV with 3H-TdR 9 c. Purification of MCMV 9 d. Infection of mouse spleen cells in vitro . . . 10 e. Plaque assays and infectious center determinations 10 f. Ultraviolet inactivation of MCMV 11 2. Mouse Embryo Cells 11 a. Culture conditions 11 b. Cell transfers 12 3. Spleen Cells 12 a. Cell preparation 12 V TABLE OF CONTENTS (Cont'd) Page b. Cell separation by nylon wool column adherence . 13 c. Cell separation by adherence to plastic tissue culture plates 14 d. Treatment of spleen cells with antiserum plus complement 14 e. Fluorescent antibody labelling of spleen c e l l s . 15 f. Non-specific esterase staining of spleen cells . 15 g. Y - i - r r a < i i a t i o n of spleen cells 16 3 h. Thymidine (methyl- H) incorporation by spleen cells 16 4. Antiserum Preparation 17 a. Rabbit anti-mouse thymocyte serum 17 b. Rabbit anti-mouse IgG serum 17 c. Preparation of crude Ig fraction from serum . . 17 CHAPTER III: SUSCEPTIBILITY OF LYMPHOID CELLS TO MCMV INFECTION IN VITRO . . 17b A. Introduction 17b B. Results 18 1. Virus uptake and the establishment of infectious centers 18 2. Rescue of MCMV from infectious centers 22 3. MCMV replication in spleen cultures 28 4. Identification of target cells for MCMV replication v i TABLE OF CONTENTS (Cont'd) Page a. Cell separation by nylon wool column adherence . 34 b. Cell separation by adherence to tissue culture plastic petri dishes 37 c. Cell separation by adherence to tissue culture dishes followed by adherence to nylon wool columns 41 d. Characterization of c e l l types by their resistance to y-xay irradiation and adherence to plastic tissue culture dishes 47 e. Characterization of c e l l types by the use of Anti-Ig serum plus complement 54 C. Discussion 54 CHAPTER IV: THE EFFECT OF MCMV INFECTION ON THE IMMUNE RESPONSE OF MOUSE SPLEEN CELLS 59 A. Introduction 59 B. Results 60 1. The effect of in vitro MCMV infection on the mitogen response of mouse spleen cells 60 2. Comparison between MCMV and UV-inactivated MCMV . . 64 3 3. H-TdR incorporation by spleen cells 66 4. Effect of changing mitogen concentration 69 5. Possible mechanisms for immunosuppression . . . . 69 C. Discussion 74 v i i TABLE OF CONTENTS (Cont'd) Page BIBLIOGRAPHY 76 v i i i LIST OF TABLES Table Page CHAPTER III: I I I - l Comparison between infectious centers (I.C.) 20 and infectious virions (p.f.u.). III-2 Correlation between virus uptake and infectious 21 center formation. III-3 Relationship between the multiplicity of 23 infection (M.O.I.) and MCMV uptake by SWR spleen ce l l s . III-4 Proportionality of multiplicity of infection 24 (M.O.I.) and infectious centers ( I . C ) . III-5 Reactivation of MCMV from spleen cells by 25 co-cultivation with syngeneic or allogeneic mouse embryo cell s . III-6 Reactivation of MCMV from infected SWR spleen 27 cells in the presence of DNase or antiserum. III-7 V i a b i l i t y of MCMV-infected and mock-infected 29 spleen c e l l s . III-8 MCMV replication in ME monolayers in the presence 32 or absence of con A (5 yg/ml). III-9 MCMV replication in nylon-wool column separated 36 C3H/HeJ spleen c e l l fractions. 111-10 Infectious center (I.C.) assays on MCMV infected 39 SWR spleen cells separated by plastic adherence. I I I - l l Infectious center (I.C.) assays on infected SWR 42 spleen cells separated by adherence to plastic tissue culture dishes. 111-12 Infectious center (I.C.) assays on infected SWR 45 spleen cells separated by adherence to plastic tissue culture dishes followed by adherence to nylon wool columns. 111-13 MCMV replication in SWR spleen c e l l fractions 46 separated by adherence to plastic tissue culture dishes followed by adherence to nylon wool columns. ix LIST OF TABLES (cont'd) Table Page CHAPTER III: 111-14 Vi a b i l i t y of SWR spleen cells after 2000 R of 48 y-irradiation. III- 15 The effect of y _ : L r r a d i a t i o n on the establish- 53 ment of infectious centers. CHAPTER IV: IV- 1 The effect of MCMV infection on the 3H-TdR 61 Uptake of SWR spleen cells. Effect of MCMV infection or of unstimulated spleen cells. IV-2a on the 3H-TdR uptake 62 3 IV-2b Effect of antiserum treatment on the H-TdR 63 uptake of spleen c e l l s . IV-3 The effects of MCMV and UV-inactivated MCMV on 65 the mitogen responses of SWR spleen c e l l s . IV-4 The effect of mitogen concentration on the 70 %-TdR incorporation of MCMV infected and normal spleen c e l l s . IV-5 The effect of MCMV infected plastic adherent 72 cells on the ^ H-TdR incorporation of mitogen stimulated SWR spleen cel l s . IV-6 The effect of MCMV infected plastic adherent 73 cells on the %-TdR incorporation of mitogen stimulated SWR spleen c e l l s . X LIST OF FIGURES Figure Page CHAPTER III: I l l - l a V i r a l replication in SWR mouse spleen cells 30 incubated with mitogens after MCMV infection in vitro. I l l - l b V i r a l replication in SWR mouse spleen cells 30 incubated with mitogens for 2 days before MCMV infection i n vitro. III-2 Murine cytomegalovirus replication in SWR 33 mouse spleen cultures in RPMI 1640 medium supplemented with 10% (v/v) untreated fetal calf serum or heat inactivated fetal calf serum. III-3 Murine cytomegalovirus replication in C3H/HeJ 35 mouse spleen cells separated by a nylon wool column after MCMV infection in vitro. III-4 Murine cytomegalovirus replication in SWR 38 mouse spleen cells separated by adherence to Falcon tissue culture dishes after MCMV infection in vitro. III-5 Murine cytomegalovirus replication in SWR 44 mouse spleen cells separated by adherence to Falcon tissue culture dishes and nylon wool columns after MCMV infection in vitro. III-6a & b Murine cytomegalovirus replication in SWR 50/51 mouse spleen cells separated by their resistance to Y ~ i r r a d i a t i o n and adherence to Falcon tissue culture dishes. CHAPTER IV: IV-1 The response of mock infected and UV-inactivated 67 MCMV infected SWR spleen cells to con A stimulation. IV-2 The response of mock infected and MCMV infected 68 SWR spleen cells to con A stimulation. ACKNOWLEDGEMENTS The author wishes to express his gratitude to Dr. James B. Hudson for his advice and guidance. Moreover, the project might not even have been started i f i t were not for his trust in me. A lot of thanks also go to Jessyca Maltman who tirelessly prepared the thousands of mouse embryo plates for my experiments and has to patiently endure my long-winded mutterings from time to time. The helpful suggestions by the advisory committee are hereby acknowledged. I am grateful to Rosemary Morgan for her s k i l f u l typing of my thesis and Chris Irving for drawing up the figures so beautifully. In addition, I like to thank the "coffee room gang" on the third floor and Sue Bergman and Pat Grass in particular, for i t i s their warmth and sensitivity that has kept my spirits up during d i f f i c u l t times. My most heartfelt thanks go to Bach, Beethoven, Mozart and Schubert. If l i f e were indeed like a play, nobody could have described my experiences in the thirty odd years of my existence than the grief-stricken Canio in his soliloquy in "I Pagliacci": Recitar! Mentre preso dal d e l i r i o Non so piu quel che dico e quel che faccio! Eppur ... e~ d'uopo ... sforzati! Ridi Pagliaccio, e ognun applaudira*! Tramuta in lazzi lo spasmo ed i l pianto; In una smorfia i l singhiozzo e i l dolor ... Ridi Pagliaccio, sul tuo amore infranto! Ridi del duol che t'avvelena i l cor! In a l i f e f i l l e d with bitter disappointments and personal tragedies, when the w i l l to go on was crushed again and again by fate, i t is music, so human and beautiful, that has consoled and encouraged me. Who would have remained unmoved by the heroism and tragedy in Beethoven's "Eroica" or Mozart's G-minor symphonies? Then there is the serenity and sad resignation of Schubert's great C major quintet. The l i s t goes on and on. In a near fa t a l accident when injustice and personal loss have combined to drive me to the brink of disaster, i t i s Bach's incomparable Chaconne for solo v i o l i n that has given me the strength to complete the last experiments of this project. Thus I thank a l l my friends who have performed with me at one time or another and especially B i l l Walley, members of the Purcell string quartet, Sue Round and Rina Schuurman, who shared my most cherishable moments. 1 CHAPTER I INTRODUCTION The study of the interactions between viruses and lymphoid cells has always been a subject of great interest to scientists working to control v i r a l diseases and to further our understanding of the immune system. An enormous amount of work has already been done with leukocyte cultures and different viruses, adding to our knowledge of v i r a l immunology. Viruses interact with lymphocytes in a variety of ways. Some, like the yellow fever virus, are capable of spontaneous replication in unstimulated lymphocyte cultures (Denman et a l , 1974). Others, like Vesicular Stomatitis Virus (VSV) and Herpes Simplex Virus (HSV), replicate preferentially in mitogen stimulated leuko-cyte cultures (Nahmias et a l , 1964; Edelman et a l , 1968; Kleinman et a l , 1972; Kirchner et a l , 1976), suggesting that only lymphocytes undergoing DNA synthesis can support v i r a l replication in such cases. On the other hand, certain viruses (influenza and Sendai viruses) are inactivated by a minor population of lymphocytes possibly as a result of an incomplete cycle of replication within these cells (Zisman and Denman, 1973). Moreover, lymphoid cells may actually harbor the v i r a l genome in a latent state. For example, latent Murine Cytomegalovirus (MCMV) can be recovered from spleen cells of chronically infected mice by co-cultivation with susceptible mouse embryo fibroblasts (Henson et a l , 1972; Olding et a l , 1975). Even more intriguing i s the fact that human lymphocytes, which normally do not persist in culture, can be transformed into permanent 2 cultures of lymphoblastoid c e l l lines after exposure to Epstein-Barr Virus (EBV) (Pagano, 1975). Viral infections almost invariably e l i c i t an immune response from the lymphocytes in the form of antibody production and/or virus-specific cytotoxic T-lymphocyte response (Quinnan et a l , 1978). As the infection takes i t s course, virus-induced immunosuppression may set in, impairing both cell-mediated as well as humoral immune responses (Howard and Najarian, 1974; Howard et a l , 1974) possibly as a result of v i r a l replication in and subsequent destruction of responding cel l s , or redirection of the infected lymphocyte's metabolic activity towards the production of v i r a l products. The purpose of the present project was to study the interactions between murine cytomegalovirus (MCMV) and lymphoid cells in vitro. Our experimental work was focussed on the susceptibility of lymphoid cells to MCMV infection, the c e l l types involved and the alteration of mitogen responses as a result of infection in vitro. Murine cytomegalovirus has been classified with the herpes virus family. It has a central core of double stranded DNA and an enveloped capsid consisting of 162 capsomers arranged in icosahedral symmetry. The virion can be present in a single or multicapsid form. In the latter, a collection of 1 to 20 capsids are embedded in a dense staining matrix surrounded by a single envelope, thus constituting only one infectious unit (Hudson et a l , 1976). The multicapsid virion i s the predominant form in tissue culture passaged preparations of MCMV. The virus possesses a large genome with a molecular weight of 132 x 10 daltons (Mosmann and Hudson, 1973) 3 and in i t s single capsid form, the virion has a diameter of 150-170 nm. Unlike other herpes viruses, MCMV infectivity i s considerably enhanced by centrifugation during i t s adsorption period (Osborn and Walker, 1968; Hudson et a l , 1976). During the normal v i r a l replication cycle in 3T3 cells , infectious virus particles became detectable 12 hours post infection and i n i t i a t i o n of v i r a l DNA synthesis required events associated with the host S-phase (Muller and Hudson, 1977). In mice infected with MCMV, v i r a l replication was detectable in the liver , lung, kidney and spleen up to 30 days post infection and considerably longer in the salivary glands (Kelsey et a l , 1977), until the degeneration of the infected cells and healing occurred between the sixth and ninth weeks (Henson and Strano, 1972). However MCMV could be recovered from spleen cells of infected animals as late as 5 months after infection by co-cultivation with allogeneic mouse embryo fibroblasts (Olding et a l , 1975). Mitogen responses of the lymphocytes were suppressed during the acute infection phase but recovery occurred in sub-lethal cases (Kelsey et a l , 1977). T-lymphocytes (Starr and Allison, 1977) and macrophages (Selgrade and Osborn, 1974) were implicated in resistance to MCMV infection. Murine cytomegalovirus has been chosen for study because of the similarity to human cytomegalovirus infections. The latter are widespread and play a significant role in causing human birth defects, tissue injury and in some cases, death within a few years of birth (Weiler, 1971). Reactivation of either virus can occur after blood transfusions (Cheung and Lang, 1977). The study of 4 MCMV infection w i l l also help to shed light on the general problem of virus latency and reactivation. Moreover, immunological and virological manipulations are relatively simple in mice, and inbred strains of mice are available for studying the effect of allogeneic stimulation on virus reactivation. An in vitro system has been selected because i t would allow us considerably more f l e x i b i l i t y in designing experiments. In addition, c e l l separation and other manipulations can be performed and target cells of v i r a l infection identified more easily than i f l i v e animals are used. As a bonus, in vitro infection yields a larger number of infected cells with a given dose of virus. The spleen was used as a source for lymphoid cells because MCMV has previously been recovered from i t (Henson et a l , 1972; Olding et a l , 1975). Hopefully, the experimental results presented in the following chapters w i l l be the prelude to the establishment of an in vivo experimental system. 5. CHAPTER II MATERIALS AND METHODS Materials 1. Reagents: Name Agarose Alpha-naphthyl acetate Complement (guinea pig) Concanavalin A Deoxyribonuclease (RNase free) Ethylene Glycol Monomethyl Ether F.I.T.C. Conjugated Goat Anti-Rabbit IgG Freund's Adjuvant (complete) Gamma Globulins Fr.II (mouse) Gentamycin IgG Fraction Rabbit Anti-Mouse Gamma Globulin IgG Fraction Rabbit Anti-Mouse Thymocytes Lipopolysaccharide (S. typhimurium) Mycostatin Pararosanilin Penicillin-Streptomycin-Fungizone Ribonuclease A Sodium Nitrite Spectrafluor Thymidine (Methyl-3H), 47 Ci/mmol. Trypan Blue Trypsin Source Seakem Bausch and Lomb Sigma Chemical Co. Miles Laboratories Inc. Sigma Chemical Co. Sigma Chemical Co. Sigma Chemical Co. Miles Laboratories Inc. Difco Miles Laboratories Inc. Sigma Chemical Co. Cappel Laboratories Inc. Cappel Laboratories Inc. Difco Difco Sigma Chemical Co. Gibco Sigma Chemical Co. Sigma Chemical Co. Amersham Searle Co. Amersham Searle Co. Matheson, Coleman and Bell. Difco A l l other reagents used were from Fisher Scientific Chemical Co. Solutions and Buffers: Buffered Formalin Acetone Fixative (pH 6.6) Na2HP04 20 mg KH2P04 100 mg Acetone 45 ml Formalin 25 ml Water 30 ml Hanks' Balanced Salt Solution (Hanks' BSS) Glucose 5.5 mM NaCl 147.0 mM KC1 5.3 mM KHJPO. 4.4 mM 2 4 Na_HP0. 15.0 mM 2 4 Phenol Red 0.6 mM Phosphate Buffered Saline (PBS) NaCl 130.0 mM KC1 2.7 mM NaHPO. 8.1 mM 2 4 KHoP0. 1.5 mM 2 4 CaCl 2 1.0 mM MgCl 2 0.5 mM Scin t i l l a t i o n Fluid Spectrafluor 42 ml per l i t e r of Toluene 7 Trypan Blue Solution Trypan Blue 0.7 gm per l i t e r NaCl 8.5 gm per l i t e r 3. Growth media: a. Mouse Embryo Monolayers. Eagle's minimal essential medium (MEM), Dulbecco's modification, was used. The MEM was obtained from Gibco in powdered form, rehydrated and f i l t e r s t e r i l i z e d . For cultivation i n petri dishes i n a 37°C CO2 incubator, 3.7 g/1 NaHCO^ were added (MEM-A), but for cultures kept in capped r o l l e r bottles in a 37°C incubation room, 1.5 g/1 NaHCO^ were added (MEM-B). In addition, antibiotics were routinely added to the media after s t e r i l i z a t i o n (gentamycin, 20 yg/ml). Fetal calf serum (Gibco) was added to a concentration of 2, 5 or 10% (v/v) as required. b. Spleen Cells. Spleen cells were cultured in RPMI 1640 medium (Flow Laboratories) with 10% (v/v) fetal calf serum in polypropylene tubes (Fisher Scientific Company) kept in a 37°C CO^ incubator. The fet a l calf serum had not been heat inactivated unless indicated otherwise. B. Methods 1. Viruses: The Smith strain of MCMV, from the American Type Culture Collection, was used i n these studies. 8 a. Growth of MCMV Murine cytomegalovirus was propagated in tertiary or quaternary mouse embryo (ME) cel l s . For large scale production of MCMV, roller bottle cultures of ME cells were infected at low multiplicity (0.1 or less pfu/cell). After a one hour adsorption period, cultivation was continued in MEM-B plus 2% (v/v) fetal calf serum at 37°C unt i l over 90% of the cells showed cytopathic effect. MCMV was then purified from the supernatant medium by differential centrifugation. For MCMV propagation in ME cells grown in petri dishes, either standard or centrifugal infection was used. (i) Standard infection Medium was drained from the cultures and a minimal volume (1.0 ml for 35 mm dishes, 2.0 ml for 50 mm dishes and 4.0 ml for 90 mm dishes) of MEM-B plus 2% (v/v) fetal calf serum containing MCMV was added. After a one hour adsorption period at 37°C, the inoculum was removed and the dishes were given pre-warmed medium and placed in a 37°C CO2 incubator. ( i i ) Centrifugal infection Centrifugal infection was essentially the same as above except that during the adsorption period the petri plates (35 mm or 50 mm plates) were stacked in the buckets of an IEC model CS centrifuge and centrifuged at 2,000 rpm for 30 minutes at room temperature. Then cultivation continued as above after the media change. 9 b. Labelling of MCMV with H-TdR. Medium was drained from confluent ME monolayers, in 50 mm petri plates and MCMV was added in MEM-B plus 2% (v/v) fetal calf serum at an input multiplicity of 10 pfu/cell. Centrifugal infection was carried out as outlined above. The medium in each plate was replaced with 4 ml of fresh MEM-A plus 2% (v/v) fetal calf serum containing 3 20 yCi of H-TdR 12 to 14 hours post infection. Another 20 yCi of 3 H-TdR were added to each plate 20 hours post infection. Incubation at 37°C was continued u n t i l about 40 hours post infection when over 90% of the ME cells should have shown cytopathic effect. MCMV was then purified from the supernatant by differential centrifugation and DNase treatment. c. Purification of MCMV. The media were collected from MCMV infected ME cultures and c e l l debris was removed by centrifugation at 2,000 rpm for 15 minutes in an IEC-CS centrifuge. The virus i n the supernatant was then pelleted by centrifugation at 24,000 g for 3 hours i n the GSA rotor of a Sorvall RC-2B centrifuge, resuspended in MEM-B plus 2% (v/v) fetal calf serum and divided into 1 ml aliquots prior to storage in a -70°C refrigerator. An extra step was taken in the purification of Tritium labelled MCMV. After the i n i t i a l high speed centrifugation, the virus pellet was resuspended i n MEM-B and treated with DNase (50 ug/ml) and RNase (100 ug/ml) for 15 minutes at 37°C to remove traces of cellular DNA and RNA. Then the high speed centrifugation was repeated and the 10 resulting virus pellet resuspended in MEM-B plus 2% (v/v) fetal calf serum prior to storage in the -70°C refrigerator. d. Infection of mouse spleen cells in vitro. Mouse spleen cells at a concentration of 2 x 10^ cells/ml were infected with MCMV at a multiplicity of infection of one unless otherwise indicated. After an adsorption period of one hour at 37°C, the cells were washed three times to remove unadsorbed virus, resuspended in fresh medium (RPMI 1640 plus 10% (v/v) fetal calf serum) and diluted to a concentration of 10 cells/ml for cultivation in a CO2 incubator at 37°C. Samples were taken for infectivity assays at various times after infection. e. Plaque assays and infectious center determinations. MCMV was assayed by plaque formation on freshly confluent ME monolayers, usually employing the standard infection method described previously. Cell associated virus could only be assayed after the infected spleen cells were disrupted by three cycles of freezing and thawing. After the adsorption period, the infected ME monolayers were overlaid with MEM-A containing 0.5% (w/v) Agarose and 2% (v/v) fet a l calf serum. Plaques should become evident after 4 days of incubation at 37°C. Infectious centers were assayed by plating infected spleen cells on to freshly confluent ME monolayers. Without removing the inoculum after a 60 minute adsorption period, MEM-A mixed with Agarose was added to give an overlay with a f i n a l concentration of 11 0.5% (w/v) Agarose in MEM-A. Plaques could be counted after 4 days of incubation at 37°C. A slight modification was made in infectious center assays involving plastic-adherent c e l l s . Instead of removing the adherent cells from the tissue culture plates, freshly trypsinized mouse embryo fibroblasts were added and allowed to settle for about 6 to 8 hours before the c e l l monolayer was overlaid with MEM-A containing 0.5% (w/v) Agarose and 2% (v/v) fetal calf serum. Plaques were countable after 4 days of incubation at 37°C. In a l l cases, such assays were done at least in duplicate at each vi r u s / c e l l dilution. The plaque counts in each set of duplicates rarely differed by more than 10%. f. Ultraviolet inactivation of MCMV. The virus preparation, spread out in one or more open petri dishes, was irradiated by UV light from a 30 watt germicidal lamp at a distance of 16 cm for 5 minutes. The virus infectivity was found to be reduced at least by a factor of 10 . 2. Mouse embryo cells (ME) Mouse embryo cells were derived from embryos of random bred Swiss white mice unless otherwise indicated. a. Culture conditions Cells grown on 35 mm, 50 mm, or 90 mm Falcon tissue culture dishes (Fisher Scientific Company) were incubated in a humidified atmosphere containing 5% C0 9 and 95% air at 37°C. Cells grown in capped r o l l e r bottles were incubated at 37"C on a Bellco c e l l production rol l e r apparatus. b. Cell transfer. Mouse embryo cells were usually subcultured at confluence. The medium was decanted and the monolayers were gently rinsed once with pre-warmed Hanks' BSS to remove debris. Trypsin (0.25% in Hanks' BSS) was then added for 7 minutes and decanted. The cells were resuspended in a small volume of fresh MEM-A plus serum. Cell clumps were broken up by repeated pipetting. The c e l l suspension was then diluted to the appropriate density and dispensed into fresh culture vessels for further incubation. 3. Spleen c e l l s . The following strains of mice were the sources of spleen cells used in the experiments: SWR, C3H/HeJ and random bred Swiss white. a. Cell preparation Spleen cells were obtained by teasing the cells out of the bisected spleen into RPMI 1640 medium (with 10% (v/v) fetal calf serum). The cells were pelleted by centrifugation at 1,000 rpm for 5 minutes in the IEC-CS centrifuge, washed once with medium and resuspended in 1/6 M - NH^Cl for 4 minutes. The cells were then pelleted as before, washed once, and resuspended in the culture medium. In most experiments, after MCMV infection and c e l l sep-aration procedures were completed, the spleen cells were diluted to a concentration of 10° cells/ml and distributed into polypro-pylene tubes for incubation at 37°C in a CC^ incubator. Cell v i a b i l i t y was monitored by counting trypan blue excluding cells in a hc.emocytometer at various times throughout each experiment. b. Cell separation by nylon wool column adherence. A 0.6 gm amount of nylon wool from a LP-1 Leuko Pak (Fenwal Laboratories) was soaked in d i s t i l l e d water for one week with daily water changes. Then the nylon wool was dried, packed into a 10 ml plastic syringe up to the 8 ml mark and sterilized by autoclaving. Before adding the spleen c e l l s , the column was f i l l e d with RPMI 1640 medium plus 10% (v/v) fetal calf serum and incubated o 8 for 30 minutes at 37 C. Then a maximum of 10 cells in 1 ml of medium were added to the column and incubation continued for 45 minutes at 37°C. Then non-adherent ce l l s were eluted with about 15 ml of culture medium at a rate of one drop every 2 seconds. The adherent cells could be removed by adding fresh medium to the column, loosening the adherent cells by squeezing the nylon wool with a pair of sterile forceps and collecting the eluted c e l l s . In every case, the v i a b i l i t y of both c e l l fractions was over 90% as determined by trypan blue exclusion. The percentage of spleen cells that were nylon wool adherent was found to be about 50% for SWR mice and between 60 and 65% for C3H/HeJ mice. Compared to the unfractionated spleen c e l l s , the percentage of surface Ig bearing cells in the adherent population was enriched by a factor of two. 14 G . C e l l separation by adherence to plastic tissue  culture plates. Spleen cells in RPMI 1640 medium plus 10% (v/v) fetal calf serum at a concentration of 5 x 10 cells/ml were added to 50 mm Falcon tissue culture plates (2 ml/plate), or Linbro microtiter plates (0.2 ml/well), and incubated i n a CO^ incubator for two hours at 37°C. Non-adherent cells were removed by washing the plates vigorously three times with fresh culture medium, pelleted by centrifugation and resuspended in culture medium for further incubation. RPMI 1640 medium plus 10% (v/v) fetal calf serum was also added to the plates i f further incubation of the adherent cel l s was desired.. Using Non-Specific Esterase staining as a means of identification, over 50% of the plastic adherent ce l l s could be classified as macrophages. d. Treatment of spleen cells with antiserum plus  complement. Spleen cells were pelleted by centrifugation, washed once with Hanks' BSS, and resuspended in an equal volume mixture of guinea pig complement and rabbit anti-mouse IgG or rabbit anti-mouse thymocyte serum at dilutions previously found to give maximum c e l l k i l l i n g . The mixture was incubated at 37°C in a CO^ incubator for 60 minutes. The spleen cells were then pelleted again by centrifugation, washed once with Hanks' BSS and resuspended in culture medium for further incubation. In the case of SWR spleen cell s , about half the cells were k i l l e d after such treatment and roughly two-thirds of the surface Ig bearing cells were eliminated in the process. 15 e. Fluorescent antibody labelling of spleen c e l l s . Spleen cells were pelleted by centrifugation, washed once with PBS and incubated with rabbit anti-mouse IgG serum (1:32.. dilution) at 4°C for 30 minutes. Then the cells were washed three times with PBS and incubated with F.I.T.C.-conjugated goat anti-rabbit IgG gamma globulin (1:16 dilution) at room temperature for 30 minutes. Finally, after thorough washings with PBS, the cells were resuspended in a 1:1 (v/v) mixture of glycerol and PBS and mounted on glass slides for observation under a Leitz fluorescence microscope. f. Non-Specific Esterase staining of spleen cel l s . The method of Yam, L i and Crosby (Yam et a l . , 1971) was used. Cell smears were fixed i n a buffered formalin-acetone fixative for 30 seconds at 4 to 10°C, washed by three changes of d i s t i l l e d water and air-dried at room temperature for 30 minutes. The fixed smears were then incubated for 45 minutes at room temperature i n the following medium: Phosphate buffer (M/15, pH 7.6) 8.9 ml Hexazotized pararosanilin 0.6 ml Alpha-naphthyl acetate 10 mg per 0.5 ml ethylene glycol monoethyl ether. The pararosanilin solution was prepared by mixing an equal volume of the pararosanilin solution and a fresh 4% sodium n i t r i t e solution for 1 minute before use. The f i n a l pH of the incubating medium was adjusted to pH 6.1 with IN NaOH and filtered before use. After the incubation, the c e l l smears were washed with water, 16 counterstained with 1% methyl green for 1 to 2 minutes, washed with water again and air dried before examination under a light microscope. Enzymic activity was seen as dark red granules in the cytoplasm of monocytes and macrophages. g. y-irradiation of spleen c e l l s . 60 A Co y-ray source i n the Chemistry Department was used. Spleen cells in polypropylene tubes were given 2000 R of y-irradiation by lowering the y-ray source into the shielded chamber where the cells were placed for 62.5 seconds. 3 h. Thymidine (methyl- H) incorporation by spleen c e l l s . Spleen cells at a concentration of 5 x 10^ cells/ml were dispensed into the wells of Linbro microtiter plates (0.2 ml/well) and mitogens were added to give f i n a l concentrations of 5 yg/ml for con A or 40 yg/ml for LPS. In the c e l l mixing experiments described in Chapter IV, i t was assumed that approximately 10% of the spleen ce l l s were plastic-adherent, thus 0.2 mis of non-adherent cells at a concentration of 4.5 x 10 cells/ml were added to the adherent cells in each well prior to mitogen addition. Thymidine incorporation of the spleen cells was monitored by incubating these microtiter cultures with H-TdR (2.5 yCi/well) for 12 hours, usually from 24 to 36 hours as well as from 60 to 72 hours after mitogen addition. The cells were then harvested by a custom made harvester and dried on fiberglass f i l t e r s . In most cases, i n order to get a better signal to noise ratio, trichloroacetic acid (TCA) precipitation was carried out at 4°C for 60 minutes by soaking the f i l t e r s in cold 10% (w/v) TCA. Then the f i l t e r s were washed twice with 5% (w/v) TCA, once with ethanol and dried before radioactivity was measured in a s c i n t i l l a t i o n counter. 4. Antiserum preparation. The rabbit anti-mouse IgG and rabbit anti-mouse thymocyte sera used i n the early experiments were prepared in our laboratory. In later experiments, they were obtained commercially. The rabbit anti-MCMV serum was kindly donated by Dr. Vikram Misra. a. Rabbit anti-mouse thymocyte serum The method described by Golub (Golub, 1971) was used. The brain of a Swiss white mouse was removed and homogenized in 0.5 ml of Hanks' BSS. The resulting suspension was emulsified with an equal volume of Freund's complete adjuvant and injected intramuscularly into a New Zealand white rabbit. The injection was repeated after two weeks and the rabbit was bled ten days after the second injection. b. Rabbit anti-mouse IgG serum The procedure was the same as above except mouse IgG (1 mg/ml) was used instead of mouse brain. c. Preparation of.crude Ig fraction from serum The blood from the rabbit was allowed to clot overnight at 4°C. The serum fraction was obtained by removing the clot and collecting the supernatant after one low speed centrifugation. The rabbit anti-mouse IgG serum was adsorbed against mouse thymocytes (5:1 v/v) and the rabbit anti-mouse thymocyte serum against homogenized mouse liver (5:1, v/v) for 30 minutes at 4°C before the next step. After the adsorption process, saturated ammonium sulfate solution was added dropwise to the serum with vigorous st i r r i n g u n t i l a 40% (v/v) mixture was obtained. After allowing the mixture to settle overnight at 4°C, i t was centrifuged at 15,000 g for 15 minutes and the precipitate was resuspended in Hanks' BSS or PBS. The precipitation process could be repeated a second time. CHAPTER III SUSCEPTIBILITY OF LYMPHOID CELLS TO MCMV INFECTION IN VITRO. A. Introduction. The work of several laboratories has already established the fact that MCMV can be recovered from spleen cells of latently infected mice. Gardner's group (Gardner et a l , 1974) administered anti-thymocyte serum to wild mice, while Jordan (Jordan et a l , 1977) used a combination of anti-lymphocyte serum and cortisone acetate treatment on mice subcutaneously inoculated with MCMV. Both groups showed that virus could only be isolated from the splenic tissue of mice receiving immunosuppressive treatment. Henson (Henson et a l , 1972) and Olding (Olding et a l , 1975) demon-strated that MCMV could be reactivated from whole spleen cells from latently infected mice by co-cultivation with mouse embryo fibroblasts. In a more recent report, Cheung (Cheung et a l , 1977) was also able to demonstrate MCMV reactivation in latently infected mice that were recipients of blood transfusions from uninfected donors. Thus i t is evident that splenic lymphocytes can harbor the v i r a l genome in a latent state and .that under certain conditions such as immunosuppression or antigenic stimulation, lymphoid cells are capable of supporting MCMV replication. However, because of the small number of lymphocytes involved, there was no conclusive evidence of the actual c e l l type involved in v i r a l replication and latency.• It was the objective of this project to define the conditions under which MCMV reactivation could occur and to identify the c e l l types involved by varying culture conditions and employing c e l l separation techniques. B. Results. 1. Virus uptake and the establishment of infectious centers. F i r s t , the term infectious center (I.C.) has to be defined so that the experimental results can be expressed quantitatively in a meaningful manner in the majority of cases. If N represents the number of plaques formed on co-cultivation of infected spleen cells with mouse embryo fibroblasts (MEF) and V, the number of c e l l associated and extracellular infectious v i r a l particles in the sample, then, The number of I.C. = N - V Since more than one infectious virus particle can become associated with a single c e l l , the number of infectious centers expressed in this way would represent the minimum number of cells harboring the v i r a l genome in a non-infectious state at the time of sampling. Thus each infectious center can be either a c e l l in which the v i r a l replication cycle i s passing through the eclipse period when no infectious virus particles are detectable, or one harboring MCMV in a true latent state from which reactivation can occur only under suitable conditions. 19 At most times, with efficient c e l l washing before I.C. assays to remove extracellular virus, the measured number of infectious centers is a positive number, the exception being the period of peak virus production when the number of cell-associated virus particles per c e l l becomes so great that the equation for I.C. measurement is no longer valid. Table I I I - l illustrates that the above way of measuring the number of infectious centers i s indeed a valid one. Here i t was also shown that only infectious MCMV particles and not infectious centers were subject to centrifugal enhancement of infectivity (Osborn and Walker, 1968). The small c/s ratio in the I.C. assay indicated that the author was indeed measuring the number of intact cells carrying the virus intracellularly, and that the washing had removed close to 100% of the extracellular virus. The infectious virion assay for an identical sample where the cells had been disrupted by freezing and thawing had the normal c/s ratio and represented the number of residual virus particles after washing. When spleen cells from random bred or inbred mice such as C3H and SWR strains were exposed to MCMV, a small percentage of the input virus was taken up by the cells and an even smaller percentage (usually <1% at a multiplicity of infection of 1) of the exposed lymphoid cells was able to form infectious centers upon co-cultivation with mouse embryo monolayers. Table III-2 illustrated the fact that even though a similar percentage of infectious and inactivated virus particles was taken up by the cells shortly after the adsorption period, infectious centers Table I I I - l : Comparison between infectious centers (I.C.) and infectious virions (p.f.u.). Mode of infection I.C./ml p.f.u./ml Standard (S) 6.75 x 10 3 3.3 x 10 2 Centrifugal (C) 1.67 x 10 4 8.65 x 10 3 Ratio (C/S) 2.5 26.2 21 Table III-2: Correlation between virus uptake and infectious center formation. Virus M.O.I. I.C./10 cells Cell Associated Hours Radioactivity Post-Infection MCMV 1 3.6 x 10 1.7 2 UV-MCMV* 1 <3 2.0 2 * UV-MCMV represented a MCMV sample that had been irradiated with ultraviolet light to effect at least a 10 -fold reduction in infectivity t i t e r although the number of virus particles/sample remained the same. t % of input radioactivity that was taken up by the SWR spleen c e l l s . 22 were established only in spleen cells exposed to infectious MCMV. As the multiplicity of infection (M.O.I.) was increased, the amount of virus taken up by the cells also increased. This i s illustrated in Table III-3 where 3H-TdR labelled MCMV was used to measure the amount of virus uptake. The number of infectious centers established during in vitro infection also increased with increasing multiplicities of infection. This i s shown in Table III-4. However, even at a multiplicity of infection of 25, infectious centers were established in less than 3% of the cells. Thus either the activation of MCMV by co-cultivation with mouse embryo (ME) monolayers i s extremely inefficient or only a small sub-population of the spleen cells i s capable of forming infectious centers. Both may be true. 2. Rescue of MCMV from infectious centers. The source of spleen cells for our experiments was usually kk o Q C3H/HeJ (H-2 ) and SWR (H-2 ) mice and infectious center assays were done routinely with Swiss white MEF, which should be allogeneic to both strains of mice used. In order to ascertain the require-ment for allogeneic stimulation in our infectious center assays, infected spleen cells were co-cultivated with either syngeneic or allogeneic MEF monolayers. The results from one such experiment are shown in Table III-5. It i s evident that syngeneic and a l l o -geneic MEF monolayers are equally efficient in promoting infectious center formation. Table III-3: Relationship between the multiplicity of infection (M.O.I.) and MCMV uptake by SWR spleen ce l l s . M.O.I. % Cell-Associated CPM Equivalent p.f.u. (pfu/cell) uptake/106 cells 1 0.61 6.1 x 10 10 0.29 2.9 x 10 4 100 0.19 1.9 x 10 5 24 Table III-4: Proportionality of multiplicity of infection (M.O.I.) and infectious centers ( I . C ) . M.O.I, (pfu/cell) I.C./106 cells 25 2.70 x 10 4 2.5 1.45 x 10 3 0.25 72 0.025 3.5 0.0025 <1 Table III-5: Reactivation of MCMV from spleen cells by co-cultivation with syngeneic or allogeneic mouse embryo cell s . Source of infected Source of ME I.C. per 10 spleen cells cells spleen cells SWR Swiss+ 3.4 X 10 3 SWR SWR 3.4 X 10 3 SWR C3H 3.0 X 10 3 C3H Swiss 4.3 X 10 3 C3H SWR 6.4 X 10 3 C3H C3H 4.0 X i o 3 + Random bred Swiss white mice. The formation of plaques in the MEF monolayer upon co-culti-vation with infected spleen cells required either the transport of infectious MCMV DNA into the ME cells from the spleen c e l l s , or the spread of infectious virus particles to their neighbouring ME cells after v i r a l replication in the infected spleen c e l l s . If the former were true, then at the time of actual transmission, the number of plaques in the ME monolayer would be reduced by the presence of DNase and not by anti-MCMV serum whereas the reverse should happen for the alternate mechanism. Thus experiments were performed in which at hourly intervals after the start of co-cultivation, duplicate samples were treated with either anti-MCMV serum, DNase or medium alone for 60 minutes and washed thoroughly before the addition of an agarose overlay which did not contain antiserum or DNase. The results from two experiments were shown in Table III-6. In experiment 1, data were actually available for the 72 hour period following the i n i t i a t i o n of co-cultivation. However the ratios ^Antiserum and ^DNase from the omitted time periods did not differ significantly from unity. It is interesting to note that in both experiments the only significant deviations from unity occur in the values of antiserum around the 49th to 50th hour period although co-cultivation was initiated at different times after infection in the 2 experiments. Thus the results not only show that MCMV did replicate in the spleen cells harboring the v i r a l genome and emerged as infectious virus particles to infect the neighboring ME ce l l s , they also suggest that the i n i t i -ation of v i r a l replication in infected spleen cells may require contact with ME cells at least in some cases. Table III-6: Reactivation of MCMV from infected SWR spleen cells in the presence of DNase or antiserum. Time after co-cultivation (hrs.) DNase Antiserum Expt. 1. (Co-cultivation initiated 24 hours after infection). 46 1.07 1.08 47 0.92 1.07 48 1.10 1.01 49 1.13 0.44 50 0.96 0.56 51 1.16 0.89 52 1.12 0.87 53 1.06 0.97 i-cultivation initiated 11 hours after infection) 46 1.02 0.91 47 0.94 0.87 48 0.92 0.90 49 0.87 0.83 50 0.87 0.56 51 1.03 1.00 52 0.99 0.95 53 1.08 0.87 * ^Wase = ratio: I.C. with DNase treatment/I.C. without DNase t Antiserum = ratio: I.C. with antiserum treatment/I.C. without antiserum. 1/8 dilution of rabbit anti-MCMV serum was used. 3. MCMV r e p l i c a t i o n i n spleen cultures. Spontaneous MCMV r e p l i c a t i o n did occur i n spleen cultures (both C3H/HeJ and SWR spleens) infe c t e d i n v i t r o . The vi r u s y i e l d d i f f e r e d i n d i f f e r e n t experiments probably because of differences i n s t r a i n , age or state of health of the mice used. The v i r a l r e p l i c a t i o n cycle passed through an e c l i p s e period a f t e r the ad-sorption period and an increase i n v i r u s t i t e r was observed a f t e r about 1 day i n •••.culture. The peak of vi r u s production was normally reached by the 3rd or 4th day of culture at which time most of the i n f e c t i o u s v i r u s p a r t i c l e s had already been released by the c e l l s into t h e i r surrounding medium. Since v i r u s production never exceeded 1 pfu/100 c e l l s , the number of c e l l s a c t u a l l y involved i n v i r a l r e p l i c a t i o n must be quite small. Thus any change i n c e l l v i a b i l i t y a f t e r MCMV i n f e c t i o n as determined by trypan blue ex-cl u s i o n would not be obvious. This i s shown i n Table III-7. MCMV r e p l i c a t i o n i n spleen cultures, i n contrast to herpes simplex v i r u s , was not s i g n i f i c a n t l y affected by the a d d i t i o n of mitogens (Con A or LPS) e i t h e r before or a f t e r i n f e c t i o n . This i s g r a p h i c a l l y i l l u s t r a t e d i n F i g . I I I - l a and b. In F i g . I I I - l b, d i f f e r e n t sets of spleen cultures were exposed to Con A (5 ug/ml), LPS C40 ug/ml), or l e f t untreated f o r 2 days before i n f e c t i n g with MCMV, and c u l t i v a t i o n continued without any mitogens i n the medium. In F i g . I I I - l a, the mitogens were added to the c u l t u r i n g medium r i g h t a f t e r MCMV i n f e c t i o n and maintained f o r the rest of the c u l t i v a t i o n period. I t i s seen that the only departure from the normal course of events occurred i n F i g . I I I - l a when Con A was Table III-7: V i a b i l i t y of MCMV-infected and mock-infected spleen c e l l s . Hours post infection Viable Cell Number/ml Mock Infected MCMV Infected 8 22 47 71 97 2.0 x 10" 1.2 x 10' 1.1 x 10" 2.2 x 10" 1.4 x 10" 2.6 x 10" 1.3 x 10" 2.7 x 10" 1.5 x 10" 1.3 x 10" 30 Fig. I l l - l a . SWR mouse spleen cells were incubated with mitogens after MCMV infection in vitro. The course of v i r a l replication was monitored by periodic infectivity assays. 0 control (regular culture medium only) • LPS (40 yg/ml) • Con A (5 yg/ml) Fig. I l l - l b . SWR mouse spleen cells were incubated with mitogens for 2 days before MCMV infection in vitro. The course of v i r a l replication was monitored by periodic infectivity assays. • control (regular culture medium only) • LPS (40 yg/ml) A Con A (5 yg/ml) 31 added after virus infection. The reduction in virus yield may be due to the presence of activated T-cells which have previously been shown to play a c r i t i c a l role in resistance to and recovery from MCMV infection (Gardner et a l , 1974; Booss and Wheelock, 1975; Starr and Allison, 1977). The possibility that the observed reduction in virus yield could be caused by direct inactivation by con A (Okada and Kim, 1972) as the virus i s being released from the c e l l has been eliminated by control experiments,in which i t was shown that the presence of con A neither inhibited MCMV plaque formation in infectivity assays nor prevented virus spread in i n -fected ME cultures. The result from the latter i s shown in Table III-8. In order to study factors affecting MCMV replication in spleen cultures, we cultivated infected spleen cells in media containing either normal fetal calf serum or serum that had been heat-treated to inactivate complement. The results are shown in Eig. III-2. The use of heat-treated fetal calf serum significantly reduced the virus yield. However, cl a r i f i c a t i o n as to whether this reduction was caused by the inactivation of complement or some other heat lab i l e serum factor w i l l have to await further investigation. 4. Identification of target cells for MCMV replication and  latency. It has already been shown in the previous sections that there are cells in the spleen culture that are capable of supporting Table 111-8: MCMV replication in ME monolayers in the presence or absence of con A (5 yg/ml). Hours post infection MCMV Titer (pfu/ml) normal medium medium + con A 1 33 80 96 1.0 x 10 2.7 x 10" 6.4 x 10 5.7 x 10 1.1 x 10 3.2 x 10-5.8 x 10 6.2 x 10 33 X _L 3 4 5 D a y s p o s t i n f e c t i o n Murine cytomegalovirus replication in SWR mouse spleen cultures in RPMI 1640 medium supplemented with 10% (v/v) untreated fetal calf serum (• •) or heat inactivated fetal calf serum ( •- - - - • ). MCMV replication either spontaneously or upon contact with MEF monolayers. Attempts made to characterize and isolate the target c e l l population by various c e l l separation techniques w i l l now be described. a. Cell separation by nylon wool column adherence. The use of the a b i l i t y of cells to adhere to nylon wool columns as a means for separating lymphocyte populations has been employed previously by different workers (Julius et a l , 1973; Trizio et a l , 1974; Handwerger et a l , 1974). It provides a simple yet effective way to separate T and B lymphocytes, with an approximately two-fold enrichment of T cells and 7- to 8-fold depletion of B cells in the non-adherent population, and a similar enrichment of B cells in the adherent population. Thus i f MCMV can replicate only in the general B c e l l population and not the T c e l l s , the virus yield from the infected non-adherent population would be significantly lower than either the adherent or the un-fractionated populations. This i s in fact what our results have shown. The data from one such experiment in which C3H/HeJ spleen cells were infected with MCMV just prior to column separation were plotted in Fig. III-3. To ensure that these results were not due to a change in the column-adherent a b i l i t y of the cells after MCMV infection, another experiment was performed in which the separation by nylon wool column was carried out prior to MCMV infection. The results are shown in Table III-9. Again, the non-adherent c e l l fraction produced a significantly lower yield 35 Fig. III-3: Murine cytomegalovirus replication in C3H/HeJ mouse spleen cells separated by a nylon wool column after MCMV infection in vitro. # - r • Unfractionated control cultures • - - - • Nylon wool column adherent c e l l fraction •A Non-adherent c e l l fraction 36 Table III-9: MCMV replication in nylon-wool column separated C3H/HeJ spleen c e l l fractions. Hours post infection MCMV ti t e r (pfu/10 cells) Unfractionated Adherent Non-Adherent cells cells cells 2 3.8 x 10 2 5.8 x 10 2 2.0 x 10 2 39 7.5 x 10 1 1.6 x 10 1 5.0 94 4.0 x 10 2 1.6 x 10 3 1.1 x 10 2 142 1.6 x 10 3 3.0 x 10 3 5.2 x 10 2 37 of virus than the others. Therefore i t is safe to conclude that the nylon wool column adherent fraction of spleen cells i s enriched in cells capable of supporting spontaneous MCMV replication. b. Cell separation by adherence to tissue culture plastic petri dishes. The a b i l i t y of macrophages to adhere to plastic tissue culture dishes has been utilized by many workers for selectively isolating them (Calderon et a l , 1975; Wing et a l , 1977; Hoessli et a l , 1977). Since the nylon-wool column adherent c e l l fraction i s ;enriched in both B cells and macrophages, the author decided to test the plastic adherent cells (enriched in macrophages) for their a b i l i t y to support MCMV replication in vitro. On the average, about 10% of the spleen cells remained adherent to plastic tissue culture dishes after a 2 hour incubation period at 37°C followed by thorough washing to remove loose cells. These were designated plastic adherent cel l s . The MCMV replication curves for the fractionated and unfractionated c e l l populations are shown in Fig. III-4. The plastic non-adherent c e l l fraction and the unfractionated c e l l population produced virus at a similar rate with the former at a slightly lower level, but the plastic adherent c e l l fraction appeared to be more restrictive to virus replication. MCMV production for this c e l l fraction peaked at 3 days post i n -fection and declined rapidly. The data for infectious center assays as shown in Table 111-10 were even more interesting. It was mentioned at the beginning of 38 D a y s p o s t i n f e c t i o n F i g . III-4: Murine cytomegalovirus r e p l i c a t i o n i n SWR mouse spleen c e l l s separated by adherence to Falcon tissue culture dishes a f t e r MCMV i n f e c t i o n i n v i t r o . • Unfractionated c o n t r o l cultures - - - • Spleen c e l l s non-adherent to Falcon tissue culture dishes --«~-"~=--A Spleen c e l l s adherent to Falcon tissue culture dishes 39 Table 111-10: Infectious center (I.C.) assays on MCMV infected SWR spleen cells separated by plastic adherence. Hours post T /-• t / n n6 i n . . I.C. s/10 cells xnfection Unfractionated Plastic Adherent Plastic Non-cells cells Adherent cells 5 8.9 x 10 2 4.4 x 10 3 1.2 x 10 3 27 1.3 x 10 2 3.8 x 10 2 7.6 x 10 1 48 2.4 x 10 2 5.0 x 10 2 2.1 x 10 2 72 8.1 x 10 2 3.8 x 10 2 5.3 x l O 2 113 1.3 x 10 3 1.4 x 10 2 6.8 x 10 2 144 1.3 x 10 3 5.5 x 10 1 8.0 x 10 2 this chapter that the infectious centers could represent cells harboring the v i r a l genome in a true latent state,or those in which v i r a l replication was passing through the eclipse period. If the majority of infected cells in a c e l l fraction belonged to the latter, a significant drop in the number of infectious centers in the population with time would be expected as the v i r a l replication cycle passed out of the eclipse period and the c e l l would no longer be counted as an infectious center. However, i f a large percentage of the infected cells were latently infected, the number of infectious centers in the population would remain relatively constant, dropping only as the c e l l v i a b i l i t y dropped during the cultivation period. Going back to the data in Table 111-10, we can see that the plastic non-adherent c e l l fraction f i t s into the f i r s t category. There was the i n i t i a l drop in the number of infectious centers due to both an i n i t i a l drop i n c e l l v i a b i l i t y and the v i r a l replication cycle passing out of the eclipse period. Then, coincident with the f i r s t release of virus particles and their subsequent infection of the neighboring susceptible cel l s , there was an increase in the number of infectious centers as these infected cells passed into the eclipse period of a new replication cycle. This continued as the virus spread throughout the culture. This is borne out in Fig. III-4 which shows a continuous release of infectious MCMV particles after the i n i t i a l rise in virus t i t e r . On the other hand, the plastic adherent cells behaved as latently infected cells would. There was an i n i t i a l drop in the number of infectious centers corres-41 ponding to the i n i t i a l drop in c e l l v i a b i l i t y . Then the number of infectious centers remained relatively constant during most of the remaining cultivation period. In a similar experiment, but with the data on infectious center assays expressed in a slightly different manner in Table I I I - l l , we can see that at the time period just prior to the f i r s t release of newly synthesized virus particles, the percentage of I.C. forming cells in the plastic non-adherent fraction i s significantly lower than the other two c e l l populations and 42% of the observed infectious centers are found in the plastic-adherent c e l l fraction, a 24% increase from the i n i t i a l percentage. One possible explanation i s that a high percentage of the i n i t i a l infectious centers in the plastic non-adherent fraction were in fact replicating virus spon-taneously and so were no longer counted as such as soon as they passed through the eclipse period. In conclusion, the data suggest that the plastic-adherent fraction of the spleen c e l l population i s more resistant to the spread of MCMV during the in vitro cultivation period and i s l i k e l y to be enriched in cells harboring the MCMV genome in a latent state. c. Cell separation by adherence to plastic tissue culture  dishes followed by adherence to nylon wool columns. In order to further characterize the cells replicating MCMV spontaneously, we decided to combine techniques described in sections (a) and (b). After sorting out the plastic adherent cells which are enriched in latently infected ce l l s , we can separate the 42 Table I I I - l l ; Infectious center (I.C.) assays on infected SWR spleen cells separated by adherence to plastic tissue culture dishes. Unfractionated Plastic-adherent Non-Adherent Hours post cells cells cells infection Total # I.C. 3.9 X i o 4 7.4 X 10 3 (18%)* 3.3 X i o 4 5 % of I.C. _9 _9 forming cells 4.2 X 10 2 5.2 X 10 2 4.5 X 10 2 5 in fraction Total # I.C. 1.1 X i o 4 3.3 X 10 3 (42%)* 4.6 X i o 3 25 % of I.C. o _ o forming cells 1.2 X 10 2 2.9 X 10 z 6.4 X 10 3 25 in fraction % of the total number of infectious centers at a given time. remaining cells according to their a b i l i t y to adhere to a nylon wool column. The resulting nylon wool column adherent fraction should be depleted in both T cells and macrophages but enriched in B cell s . The data on v i r a l replication in the different c e l l fractions are graphically represented in Fig. III-5. Again the nylon wool column adherent fraction gave the highest yield of virus whereas the non-adherent fraction produced the least amount of virus during the cultivation period. The results of infectious center assays are presented in Table 111-12. The data for the plastic adherent fraction followed the pattern established in the previous section. The 'A' fraction recorded the largest increase in the number of infectious centers during the cultivation period possibly because i t contained the largest fraction of cells capable of supporting spontaneous MCMV replication. If uninfected spleen cells were f i r s t separated into the three fractions as described above and then exposed to MCMV, the course of v i r a l replication in the different c e l l fractions as shown in Table 111-13 was again similar to that presented in Fig. III-5, with the 'NA' fraction producing the lowest virus yield. The unusually high p.f.u. values for the plastic adherent fraction early in the cultivation period may be due to the d i f f i c u l t y of washing off the residual virus particles after the virus adsorption period and has made meaningful infectious center assays impossible. To conclude this section, i t i s f e l t that the data presented Fig. III-5: Murine cytomegalovirus replication in SWR mouse spleen cells separated by adherence to Falcon tissue culture dishes and nylon wool columns after MCMV infection in vitro. Unfractionated control cultures Spleen cells non-adherent to Falcon tissue culture dishes but adherent to nylon wool columns Spleen cells non-adherent to both Falcon tissue culture dishes and nylon wool columns 45 Table 111-12: Infectious center (I.C.) assays on infected SWR spleen c e l l s separated by adherence to p l a s t i c t i s s u e c u l t u r e dishes followed by adherence to nylon wool columns. Hours post I.C.'s/lO C e l l s i n f e c t i o n — — — Unfractionated P l a s t i c Adherent A* NAt c e l l s c e l l s 7 3.1 X i o 3 7.3 x 3 10 J 4.1 X i o 3 5.9 X i o 3 25 6.8 X i o 1 N.D.+ 1.8 X i o 2 1.3 X i o 2 59 1.1 X 10 3 4.5 x 10 3 2.0 X i o 3 4.1 X i o 2 132 3.0 X i o 2 5.6 x i o 3 1.3 X i o 3 1.6 X i o 2 202 3.0 X i o 1 3.3 x i o 2 1.3 X 10 2 3.4 X i o 1 * P l a s t i c non-adherent, but nylon wool column adherent c e l l s t P l a s t i c non-adherent, and nylon wool column non-adherent c e l l s + Not determined 46 Table 111-13: MCMV r e p l i c a t i o n i n SWR spleen c e l l f r a c t i o n s separated by adherence to p l a s t i c t i s s u e c u l t u r e dishes followed by adherence to nylon wool columns. Hours post MCMV t i t e r (p.f.u./lO c e l l s ) i n f e c t i o n Unfractionated P l a s t i c Adherent A* NAt c e l l s c e l l s 2 1.5 X i o 3 8.8 X i o 4 1.8 X i o 3 1.9 X i o 3 28 1.5 X i o 2 1.0 X i o 4 1.8 X i o 2 2.1 X i o 2 76 2.5 X i o 3 1.4 X i o 2 2.0 X 10 3 1.2 X i o 3 124 3.6 X 3 i c r 1.4 X i o2 2.7 X 10 3 1.0 X 10 3 171 2.2 X i o 3 5.1 1.8 X 10 3 5.8 X i o 2 * P l a s t i c non-adherent, but nylon wool column adherent c e l l s t P l a s t i c non-adherent, and nylon wool column non-adherent c e l l s 47 here strongly suggest that the plastic adherent c e l l fraction is enriched in latently infected cells and the plastic non-adherent but nylon wool column adherent c e l l fraction i s enriched in cells capable of supporting spontaneous MCMV replication. d. Characterization of c e l l types by their resistance to y-ray irradiation and adherence to plastic tissue  culture dishes. Macrophages are known to be much more resistant to y-irradiation than B and T cell s . Thus y-irradiation of an uninfected spleen culture should leave the macrophages functional but eliminate the y-sensitive T and B cell s . After giving 2000 R of y-irradiation to a spleen culture, i t was found that the majority of cells in the culture died off rapidly (see Table 111-14) and the surviving cells did not respond to con A or LPS stimulation. With these facts in mind, an experiment was performed in which spleen cells were given a 2000 R dose of y-irradiation before infecting with MCMV, followed by separation by adherence to plastic tissue culture dishes. A control set of cultures in which only y-irradiation was omitted was kept for comparison purposes. The results for MCMV replication in the spleen cultures are shown in Figures III-6 a and III-6 b. The 'control' cultures behaved as expected. Vi r a l replication was reduced in a l l the y-irradiated cultures with the most dramatic reduction occurring in the plastic non-adherent fraction. This is to be expected because, depleted of macrophages, this fraction contains the highest percentage of y-sensitive c e l l s . 48 Table 111-14: Vi a b i l i t y of SWR spleen cells after 2000 R of y-irradiation. Viable c e l l number/ml Hours post Unirradiated y-irradiated irradiation control cells 6 t\ 0 (before irradiation) 1.0 x 10 1.0 x 10 23 2.6 x 10 5 7.3 x 10 4 70 2.0 x 10 5 9.5 x 10 4 120 2.8 x 10 5 9.0 x 10 4 167 1.5 x 10 5 7.0 x 10 4 49 Fig. III-6 a and b: Murine cytomegalovirus replication in SWR mouse spleen cells separated by their resistance to Y~irradiation and adherence to Falcon tissue culture dishes. Cultures that were not y-irradiated Q O Unfractionated control cultures Spleen cells non-adherent to Falcon tissue culture dishes A S p l e e n cells adherent to Falcon Tissue culture dishes y-irradiated cultures (before MCMV infection) 0 '• —# Unf ractionated spleen cells • • Spleen cells non-adherent to Falcon tissue culture dishes A A Spleen cells adherent to Falcon tissue culture dishes 50 I I I -Fig. I l l - 6 b 52 Turning now to the data on infectious center assays shown in Table 111-15, i t is obvious that y-irradiation has effectively abrogated infectious center formation in the unfractionated and plastic non-adherent c e l l fractions while leaving the plastic adherent fraction mostly unaffected. Since about 10% of the spleen cells are plastic adherent, one would expect that some infectious centers would be present in the unfractionated but y-irradiated spleen c e l l population at the last time period. However, the expected number of infectious centers would be near the lim i t of detection (1 infectious center/10 cells) in our experiment. To sum up, experimental results shown in this section have reinforced the conclusions drawn from previous sections. The cells in the spleen culture that are capable of supporting spontaneous MCMV replication possess B c e l l - l i k e properties such as nylon wool column adherence, plastic non-adherence and sensitivity to y-irradiation. By eliminating these susceptible cells through y-irradiation, we have effectively stopped the spread of the virus through the culture. Hence the number of infectious centers decayed to undetectable levels during the cultivation period. On the other hand, the cells that harbor MCMV in a latent state possess macro-phage-like properties. They are plastic adherent, resistant to y-irradiation and do not respond to con A or LPS stimulation. These cells are also capable of supporting spontaneous MCMV r e p l i -cation to a limited extent and are generally resistant to the spread of the virus after the i n i t i a l uptake of the inoculum. The fact that the number of infectious centers in both plastic Table 111-15: The effect of y-irradiation on the establishment of infectious centers. Hours post I.C's/10 cells infection — - — — — — — — ______ Unfractionated Plastic Adherent Plastic Non-Adherent control y-irrad. control y-irrad. control y-irrad. 6 8.8 X i o 2 3.3 x 10 2 1.3 X i o 3 1.4 X 10 3 7.6 X i o 2 4.3 x 10 2 23 5.3 X i o 1 3.1 x 10 1 2.1 X i o 2 1.4 X i o 2 3.7 X i o 1 9.4 120 1.5 X 10 3 1.7 3.1 X i o 2 2.3 X 2 10 z 7.9 X i o 2 <1 167 1.7 X 3 10 J <1 2.8 X i o 2 9.3 X i o 1 2.8 X i o 2 <1 54 adherent fractions (y-irradiated or control samples) remained relatively stable throughout most of the cultivation period only shows more convincingly that these infectious centers must represent mostly cells harboring the MCMV genome in a true latent state activated only by co-cultivation with ME monolayers. e. Characterization of c e l l types by the use of anti-Ig  serum plus complement. The use of anti-Ig serum plus complement on spleen cultures should eliminate B cells selectively because of the surface Ig that characterize these c e l l s . Attempts were made to treat SWR spleen cells with such a combination either before or after MCMV infection and the course of v i r a l replication in the surviving cells was followed. The results from preliminary experiments did not show any significant difference between the antiserum treated and control cultures. Unfortunately, on checking the efficiency of the anti-Ig serum in eliminating B cells , we found that only two thirds of the surface Ig bearing cells had been eliminated and the antiserum treated cultures s t i l l responded to LPS stimulation although to a lower extent than untreated ones. Thus there i s no conclusive evidence as to whether the spleen cells supporting spontaneous MCMV replication are surface Ig bearing cells or not. C. Discussion For the results from an in vitro system to be meaningful, i t must be shown that i t can duplicate the main features observed 55 in the in vivo situation. It has been shown that MCMV can replicate in the spleen of the infected mouse during the i n i t i a l phase of the infection of the liv e animal (Kelsey et a l , 1977). We have shown that the spleen does contain a small population of cells capable of supporting spontaneous v i r a l replication. After the i n i t i a l acute phase of infection i n the live animal, some spleen cells can harbor the v i r a l genome in a latent state without showing any c l i n i c a l evidence of MCMV infection. We have also shown that latent infection of spleen cells can be established in the i n vitro system. Moreover, as w i l l be demonstrated in the next chapter, the immunosuppressive effect of in vivo MCMV infection on spleen lymphocytes can also be duplicated i n vitro. Thus the in vitro system described here should be a valid one for studying the inter-actions between MCMV and lymphoid cells. One of the many aspects that sets MCMV infection of spleen cells apart from that of other susceptible cells like ME fibroblasts i s the inefficiency of virus uptake by these c e l l s . At multiplicities of infection higher than 1, the percentage of virus particles taken up by spleen cells had levelled off at a few percent of the input. Moreover, even at high multiplicities of infection when close to one virus particle per c e l l was being taken up, infectious centers were established i n only a few percent of the spleen c e l l population. One possible explanation for this phenomenon i s that only a small percentage of the spleen c e l l population i s susceptible to MCMV infection. Thus the virus particle w i l l have to hit the 'right' c e l l to be taken up. For example, i f 2% of the cells are susceptible, 56 then once an equilibrium condition has been reached, only one in f i f t y random collisions between a c e l l and a virus particle w i l l result i n the virus being taken up. This would also account for the small number of infectious centers observed. In addition, i t is possible that the inefficiency of virus rescue by co-cultivation with ME monolayers and the degradation of the adsorbed virus particles within some cells would reduce this number even further. Nevertheless, f i n a l confirmation or rejection of this hypothesis would have to wait for the results from autoradiography experiments using tritium labelled MCMV to determine the actual percentage of cells capable of taking up the virus particles during the adsorption period. From the accumulated experimental data, i t is now evident that the infectious centers observed represent two kinds of cells susceptible to MCMV infection - the spontaneous virus replicating c e l l and the latently infected c e l l . The former possesses B-cell like properties such as nylon wool column adherence, plastic non-adherence and sensitivity to y-irradiation. However, the fact that spontaneous MCMV replication in spleen cells i s not affected by LPS stimulation, an essential requirement for herpes simplex virus replication in B cells (Kirchner et a l , 1976), or anti-Ig serum treatment of the infected c e l l s , has made unequivocal identification of the c e l l type involved more d i f f i c u l t . One cannot exclude the possibility that spontaneous virus replicating cells may be mono-cytes. The latently infected c e l l possesses macrophage-like pro-perties such as plastic adherence and resistance to y-irradiation. 57 They do support a limited amount of spontaneous MCMV replication, but in general can only i n i t i a t e v i r a l replication upon co-cultivation with ME monolayers. The assembled virions w i l l subsequently be transmitted to the neighboring ME cells. Olding (Olding et a l , 1975) reported that reactivation of latently infected spleen cells requires allogeneic stimulation. However, our experimental results indicated that both allogeneic and syngeneic ME cells were equally efficient in MCMV reactivation from latently infected spleen ce l l s . The discrepancy may be due to the different c e l l populations involved in the two systems or the possible presence of slight antigenic heterogeneity among our inbred strains of mice (Cheung and Lang, 1977), which may be sufficient to activate MCMV replication. Finally, one is l e f t to speculate on what kind of role is being played by each c e l l type in the in vivo situation. It is quite l i k e l y that the B c e l l - l i k e spleen cells replicating MCMV in vitro are also responsible for the observed v i r a l replication in splenic tissues of MCMV infected mice. Our observation that the virus production i n spleen cultures was reduced in the presence of con A stimulated T cells also correlates with the in vivo situation where T cells play a c r i t i c a l role i n resistance to and recovery from MCMV infection (Selgrade, et a l , 1976; Starr and Allison, 1977; Quinnan et a l , 1978). Selgrade (Selgrade and Osborn, 1974) reported that macrophages are susceptible to the spread of MCMV in vitro and may only be important in the inductive phase of the host defence against MCMV challenge. Our findings are different in that the only macrophage-like cells susceptible to MCMV infection are quite resistant to the spread of the virus. In addition, they may be responsible for MCMV latency. It remains possible that the macrophages, in response to MCMV infection in vivo, may phagocytize the extracellular virus. If enough parts of the v i r a l genome escape cellular degradation and become integrated into the host genome, reactivation can occur under suitable conditions. Otherwise, by removing the virus particles from circulation and restricting v i r a l replication, macrophages can perform a protective function in MCMV infection i n vivo. CHAPTER IV THE EFFECT OF MCMV INFECTION ON THE IMMUNE RESPONSE OF MOUSE SPLEEN CELLS. A. Introduction. The effect of MCMV infec t i o n on the immune system has been investigated by many laboratories. I t has been demonstrated that T c e l l , B c e l l as well as macrophage functions are affected during the course of the infection. R.J. Howard (Howard and Najarian, 1974) reported that macrophages showed s i g n i f i c a n t l y increased rates of carbon clearance 7 to 9 days after MCMV infec t i o n . The same workers also recorded suppression of both primary and secondary immune responses to SRBC (Howard and Najarian, 1974; Booss and Wheelock, 1975). In addition, MCMV inf e c t i o n depressed the responses of lymphocytes to mitogens such as PHA (Howard et a l , 1974), LPS (Selgrade et a l , 1976; Kelsey et a l , 1977) and con A (Booss et a l , 1975; Selgrade et a l , 1976; Booss et a l , 1977). In fact, de-pression of T c e l l response to con A often preceded c l i n i c a l signs of i n f e c t i o n and correlated w e l l with the c l i n i c a l severity of MCMV inf e c t i o n . Possibly as a result of the impairment of the host immune system, MCMV infec t i o n also markedly enhanced the su s c e p t i b i l i t y of the infected mouse to b a c t e r i a l and fungal i n -fections (Hamilton et a l , 1976; Hamilton and Overall J r . , 1978). On the other hand, MCMV infec t i o n did invoke a virus s p e c i f i c cytotoxic T-lymphocyte response (Quinnan et a l , 1978) whose peak of a c t i v i t y coincided with the decline of MCMV i n f e c t i v i t y of spleen c e l l suspensions. Therefore i t i s no surprise that mice 60 deficient i n T c e l l s succumb to MCMV inf e c t i o n or reactivation more readily (Selgrade et a l , 1976; Gardner et a l , 1974). Thus i t i s important to study the mechanism of immunosuppression and to characterize the c e l l types involved. The experimental results compiled so far from our i n v i t r o system are presented i n the following sections. B. Results. 1. The effect of i n v i t r o MCMV inf e c t i o n on the mitogen  responses of mouse spleen c e l l s . 3 MCMV in f e c t i o n depresses the H-TdR uptake by unstimulated spleen lymphocytes as we l l as those stimulated by con A or LPS. These results have been tabulated i n Table IV-1. As the m u l t i p l i c i t y of i n f e c t i o n i s increased, the degree of suppression of mitogen responsiveness also increases. At the m u l t i p l i c i t i e s of in f e c t i o n used i n these experiments, the i n s i g n i f i c a n t change i n c e l l v i a b i l i t y due to MCMV inf e c t i o n cannot account for these effects. 3 The reduced H-TdR uptake by MCMV infected but unstimulated spleen c e l l s cannot be accounted for by suppression of T c e l l s or B c e l l s alone. Results tabulated i n Table IV-2a show that c e l l populations enriched i n either T or B lymphocytes are s i m i l a r l y suppressed. Results i n Table IV-2b show that the anti - 0 serum treated c e l l s are depleted of T c e l l s and the anti-Ig serum treated c e l l s are depleted of B c e l l s as judged by their mitogen responses. Table IV-1: The effect of MCMV infection on the H-TdR Uptake of SWR spleen cel l s . Mitogen M.O.I. H-TdR Uptake Stimulation Index Infected/ : (cpm) Uninfected Con A 3.5 24310 + 1087 5.7 0.13 Con A 0.35 90133 + 13490 8.5 0.47 Con A 0.035 134089 + 16494 7.3 0.70 Con A 0.0035 179194 + 19627 16.8 0.94 Con A Uninf. 190637 + 28297 9.2 LPS 3.5 3360 + 800 0.8 0.07 LPS 0.35 12458 + 154 1.2 0.27 LPS 0.035 31262 + 2333 1.7 0.68 LPS 0.0035 32649 + 4882 3.1 0.71 LPS Uninf. 46066 + 8260 2.2 None 3.5 4286 + 663 0.21 None 0.35 10602 + 2907 0.51 None 0.035 18384 + 4041 0.89 None 0.0035 10651 + 3576 0.51 None Uninf. 20720 + 4862 + Cultures were labelled in tri p l i c a t e from 48 to 72 hours after 3 mitogen addition with 1 yCi of H-TdR per well. 3 * Stimulation Index = H-TdR Uptake by mitogen stimulated cells 3H-TdR Uptake by unstimulated cells t Multiplicity of infection. Table IV-2a: Effect of MCMV infection on the H-TdR uptake of unstimulated spleen cel l s . Cell Treatment § Infected with H-TdR Uptake Infected/ (cpm) Uninfected Anti-Ig + C» MCMV 5294 + 415 0.27 I I mock 19451 + 2932 Anti -9 + C MCMV 4270 + 763 0.35 mock 12183 + 995 * c' MCMV 6219 + 771 0.32 It mock 19513 + 4205 control MCMV 8624 + 2946 0.69 n mock 12535 + 8748 t Cultures were labelled in tr i p l i c a t e from 54 to 77 hours after 3 mitogen addition with 1 uCi of H-TdR per well. * Guinea pig complement. § After treatment, c e l l concentration was adjusted to 5 x 10 cells/ml for incubation in microtiter plates. 63 Table IV-2b; E f f e c t of antiserum treatment on the H-TdR uptake of spleen c e l l s . 3 * C e l l Treatment § Mitogen H-TdR Uptake (cpm) Stimulation Index Anti-0 + C Con A 56354 + 9289 4.6 A n t i - I g + C Con A 170233 + 8124 8.8 Control Con A 114940 + 26426 9.2 Anti-0 + C LPS 84471 + 10565 6.9 Anti-Ig + C LPS 32626 + 3579 1.7 Control LPS 34000 + 8039 2.7 Anti-0 + C None 12183 + 995 Anti-Ig + C None 19451 + 2932 Control None 12535 + 8748 t Cultures were l a b e l l e d i n t r i p l i c a t e from 54 to 77 hours a f t e r 3 mitogen add i t i o n with 1 uCi of H-TdR per w e l l . 3 * Stimulation Index = H-TdR Uptake by mitogen stimulated c e l l s 3 H-TdR Uptake by unstimulated c e l l s § A f t e r treatment, c e l l concentration was adjusted to 5 x 10^ c e l l s / m l f o r incubation i n m i c r o t i t e r plates. Moreover fluorescent antibody labelling experiments showed that about two-thirds of the surface Ig bearing cells were eliminated after anti-Ig serum treatment. 2. Comparison between MCMV and UV-inactivated MCMV. Since only a small percentage of the input virus i s actually taken up by the spleen cells during the adsorption period and an even smaller number of cells replicate MCMV spontaneously in vitro, i t i s interesting to see i f UV-inactivated virus, which cannot replicate i n either ME or spleen c e l l s , can mediate the same 3 immunosuppressive effect. Table IV-3 shows the H-TdR incorporation of spleen cells at the two indicated time periods. These are only 3 partial results from an experiment measuring the H-TdR incor-poration of spleen cells for a 72 hour period following mitogen stimulation. The spleen c e l l s exposed to MCMV showed the usual depressed mitogenic response to con A and LPS. In the earlier time period, the H-TdR incorporation of the infected cells was similar to that of the mock infected ones, but this was merely a result of the higher rate of incorporation of the infected but unstwmulated c e l l s . The stimulation index indicated that they were really responding poorly to mitogen stimulation. However, exposure to UV-inactivated MCMV did not appear to imapair their mitogen responsiveness at a l l . This would seem to suggest that mere physical contacts between virus particles and spleen cells did not constitute a sufficient condition for immunosuppression. 65 Table IV-3: The effects of MCMV and UV-inactivated MCMV on the mitogen responses of SWR spleen ce l l s . 3 A Infected Mitogen Hrs After Mitogen H-TdR Incorporation Stimulation with Stimulation (C.P.M.) Index MCMV Con A 24 - 36 317770 + 20445 3.9 UV-MCMV+ Con A 324448 + 37852 13.0 MOCK Con A " 352130 + 64115 11.0 MCMV LPS » 195483 + 25527 2.4 UV-MCMV LPS 163066 + 11545 6.6 MOCK LPS " 196523 + 16717 6.0 MCMV None 81460 + 6452 UV-MCMV None 24664 + 1725 MOCK None 32917 + 4017 MCMV Con A 60 - 72 85316 + 10034 1.9 UV-MCMV Con A 274703 + 60521 14.0 MOCK Con A " 235563 + 36858 3.4 MCMV LPS » 50038 + 16039 1.1 UV-MCMV LPS 100159 + 33188 5.0 MOCK LPS 128649 + 31643 1.9 MCMV None 44355 + 8953 UV-MCMV None 19910 + 8473 MOCK None 69706 + 4546 UV inactivated MCMV 3 * Stimulation Index = H-TdR Incorporation by mitogen stimulated cells %-TdR Incorporation by unstimulated cells 3. H-TdR incorporation by spleen c e l l s . 66 Tritiated thymidine incorporation by spleen cells is normally measured about 50 to 70 hours after mitogen stimulation. However, i t i s conceivable that the mitogen unresponsiveness at this particular time period i s due to a shifting of the time at which 3 H-TdR incorporation reaches a peak for MCMV infected spleen ce l l s . 3 Thus experiments were performed in which the H-TdR incorporation of mitogen stimulated spleen cells was followed for at least 72 hours after the addition of mitogens. Figures IV-1 and IV-2 graphically depict the results from one such experiment. Again, the con A response for spleen cells exposed to UV-inactivated MCMV paralleled that of the uninfected c e l l s . However, that of MCMV infected spl-en cells started to level off about 40 hours after stimulation with con A. Thus there i s no shifting of the peak of 3 H-TdR incorporation during the time period covered by the experiment. Results qualitatively similar to these i n figures IV-1 and IV-2 were obtained for LPS stimulation (not shown). Another interesting feature occurred in the infected but un-stimulated spleen c e l l culture. Between the 20th and 40th hour 3 periods, these c e l l s showed an increased H-TdR incorporation compared to the uninfected controls. This i s also seen in Table IV-3 at the same time period and observed in some, though not a l l 3 H-TdR incorporation experiments completed up to now. Because only a small number of MCMV particles replicate in the spleen 3 c e l l s , i t i s highly unlikely that this increased H-TdR incorporation is due to v i r a l DNA synthesis. Thus the significance of this observation i s s t i l l unclear, but i t may correspond with a similar 10' 1 _L 35 51 67 83 hrs. post stimulation 99 115 Fig. IV-1; The response of mock infected and UV-inactivated MCMV infected SWR spleen c e l l s to con A stimulation Mock infected cultures: %\ Con A (5 yg/ml) stimulated O O unstimulated control UV-inactivated MCMV ..... _ Con A (5 Ug/ml) stimulated infected cultures: A- - - - A unstimulated control 19 35 51 67 83 99 115 hrs. post stimulation i i Fig. IV-2: The response of mock infected and MCMV infected SWR spl cells to con A stimulation. Mock infected cultures: •Z~.r~ — -"• Con A (5 yg/ml) stimulated O O unstimulated control MCMV infected cultures: Con A (5 yg/ml) stimulated unstimulated control 1 0 2 I L 3 i observation by R.J. Howard (Howard, Miller and Najarian, 1974) in 3 an in vivo experiment where he reported enhanced H-TdR incorpor-ation by the spleen cells of infected mice in the early phase of infection with suppression setting i n at a later time. 4. Effect of changing mitogen concentration. Immunosuppression could have resulted from the competition between virus and mitogen molecules for similar receptor sites on the c e l l or interference with the contact between mitogen molecules and the c e l l surface. By increasing the mitogen concentration to counteract such effects, we can test this hypothesis. Results from such an experiment are tablulated in Table IV-4. No such effect was observed for con A and the optimal concentration of LPS was actually lowered. Thus the suppressive effect of MCMV cannot be explained by simple competition with mitogen molecules for receptor sites. 5. Possible mechanisms for immunosuppression. Given the facts that only infectious MCMV can mediate immuno-suppression and only a small amount of virus is taken up during the adsorption period, we are l e f t with the possibility that i f the virus i s able to get into and destroy or functionally impair a small population of cells responsible for mediating mitogen responses, then we would have an explanation that could f i t in with the known facts. Since MCMV was capable of infecting certain macrophage-like 70 Table IV-4: The effect of mitogen concentration on the H-TdR incorporation^ of MCMV infected and normal spleen cells. 3 * Mitogen yg/ml Infected H-TdR Incorporation Stimulation with (C.P.M.) index Con A 100 MCMV 610 + 66 0.17 Con A 100 MOCK 1510 + 299 0.20 Con A 20 MCMV 3704 + 682 1.0 Con A 20 MOCK 12804 + 921 1.7 Con A 4 MCMV 96444 + 35231 27 Con A 4 MOCK 518103 + 63975 70 Con A 0.8 MCMV 76336 + 9621 21 Con A 0.8 MOCK 220970 + 14908 30 LPS 400 MCMV 1087 + 150 0.31 LPS 400 MOCK 7473 + 3609 1.0 LPS 160 MCMV 4931 + 870 1.4 LPS 160 MOCK 69082 + 10399 9.3 LPS 32 MCMV 7674 + 133 2.2 LPS 32 MOCK 104521 + 16538 14 LPS 6.4 MCMV 11169 + 2276 3.1 LPS 6.4 MOCK 101025 + 14910 14 None MCMV 3559 + 1081 None MOCK 7430 + 2443 tCultures were labelled in tri p l i c a t e from 60-68 hours after mitogen addition with 2.5 yCi of ^ H-TdR per well. 3 * Stimulation Index = H-TdR Incorporation by mitogen stimulated cells 3 H-TdR Incorporation by unstimulated cells 71 cells (previous chapter), i t seemed that macrophages, being able to mediate the activation of T cells by con A (Calderon et a l , 1975; Rosenstreich et a l , 1976; Wing et a l , 1977; Stoecker et a l , 1978) could be a prime candidate for this hypothesis. To this end, an experiment was performed in which MCMV infected and mock infected spleen cells were allowed to adhere to Linbro microtiter plates. The non-adherent cells, depleted of macrophages, were then collected and redistributed to the wells of the same Linbro plates to give the 4 possible combinations of adherent and non-ad-herent cells. Then the usual H^-TdR incorporation experiment was performed. The results are tabulated.in Table IV-5. They look quite promising because a small number of infected adherent cells was able to depress the con A response of a much larger population of uninfected non-adherent ce l l s . In addition, the mixture of uninfected adherent cells and infected non-adherent cells responded to con A stimulation quite well. LPS responses, not being mediated through macrophages (Lipsky and Rosenthal, 1976), were depressed when infected non-adherent cells were present. In order to perform a 'cleaner' experiment, an additional step was taken. The cells that were to provide the uninfected or mock infected adherent cells i n the experiment were Y -irradiated to eliminate contaminating T and B cells before MCMV infection. Then 3 the 4 different c e l l mixtures were again made up and H-TdR incorpor-3 ation measured. The results are shown in Table IV-6. The H-TdR incorporation of the adherent cells during the same period was less 3 than 1% of that of the mixture, thus the H-TdR incorporation 72 Table IV-5: The effect of MCMV infected plastic adherent cells on 3 the H-TdR incorporation? of mitogen stimulated SWR spleen cel l s . Adherent Non-Adherent Mitogen H-TdR Incorporation Stimulation* Cells Cells (C.P.M.) index Cultures labelled from 30-42 hours after mitogen addition: Infected Infected Con A 274285 + 17629 18 Infected Mock Con A 424756 + 30424 46 Mock Infected Con A 166340 + 21765 25 Mock Mock Con A 315577 + 68473 110 Infected Infected LPS 49642 + 13124 3.3 Infected Mock LPS 51080 + 5214 5.6 Mock Infected LPS 19796 + 4096 1.2 Mock Mock LPS 16688 + 3026 5.8 Infected Infected None 15079 + 3988 Infected Mock None 9153 + 2065 Mock Infected None 6612 + 878 Mock Mock None 2870 + 217 Cultures labelled from 60-72 hours after mitogen addition: Infected Infected Con A 198633 + 65644 16 Infected Mock Con A 334436 + 157316 39 Mock Infected Con A 288719 + 134418 156 Mock Mock Con A 848904 + 106985 309 Infected Infected LPS 8741 + 4007 0.7 Infected Mock LPS 22421 + 7300 2.6 Mock Infected LPS 2941 + 761 1.6 Mock Mock LPS 11709 + 9257 4.3 Infected Infected None 12710 + 9745 Infected Mock None 8595 + 4101 Mock Infected None 1855 + 240 Mock Mock None 2748 + 1160 t Cultures were labelled in quadruplicate with 2.5 yCi of H-TdR per well. 3 H-TdR incorporation by mitogen stimulated cells * Stimulatxon Index = — ~ H-TdR incorporation by unstimulated cells 73 Table IV-6: The effect of MCMV infected plastic adherent cells on the H-TdR incorporationt of mitogen stimulated SWR spleen c e l l s . Adherent Non-Adherent Mitogen H-TdR Incorporation Stimulation Cells Cells (C.P..M.) Index Cultures labelled 30-38 hours after mitogen addition: Infected Infected Con A 167585 + 32084 10.4 Infected Mock Con A 241216 + 32053 8.4 Mock Infected Con A 297989 + 60044 13.6 Mock Mock Con A 394339 + 34442 13.0 Infected Infected LPS 33321 + 7893 2.1 Infected Mock LPS 54659 + 11565 1.9 Mock Infected LPS 31807 + 5801 1.5 Mock Mock LPS 64420 + 6428 2.1 Infected Infected None 16149 + 2270 Infected Mock None 28565 + 1786 Mock Infected None 21910 + 1951 Mock Mock None 30378 + 7787 Cultures labelled from 64-72 hours after mitogen addition: Infected Infected Con A 136210 + 14729 4.3 Infected Mock Con A 209830 + 32528 3.9 Mock Infected Con A 198424 + 6075 7.9 Mock Mock Con A 499457 + 67300 10.5 Infected Infected LPS 69814 + 15648 2.2 Infected Mock LPS 107643 + 9526 2.0 Mock Infected LPS 54508 + 13812 2.2 Mock Mock LPS 113427 + 18706 2.4 Infected Infected None 31890 + 7568 Infected Mock None 54138 + 4030 Mock Infected None 25100 + 9852 Mock Mock None 47461 + 13576 t Cultures were labelled in tr i p l i c a t e with 2.5 uCi of H-TdR per well * Stimulation Index = H-TdR Incorporation by mitogen stimulated cells H-TdR Incorporation by unstimulated cells observed should almost be solely due to the added non-adherent ce l l s . The infected adherent cells were again able to significantly depress the con A response of the uninfected non-adherent cells to the level of the infected mixture. The incorporated counts in the case of LPS stimulation were not affected at a l l . Suppression of LPS stimulated cells occurred only when the infected non-adherent cells were present. Thus i t is very li k e l y that by infecting the macrophages, MCMV can somehow alter the cellular metabolic act i v i t i e s , thereby rendering them non-functional as a mediator for the activation of T cells by con A. The depressed response to LPS stimulation, on the other hand, i s l i k e l y to be mediated via a different route. C. Discussion We have been able to duplicate i n our in vitro system the immunosuppressive effect of MCMV infection on the responses of 3 spleen cells to con A and LPS stimulation. In addition, the H-TdR incorporation of unstimulated spleen cells i s also depressed. Increased suppression is brought on by increasing the multiplicity of infection. The observed immunosuppression is not a simple case of com-petition with mitogen molecules for receptor sites as increasing mitogen concentrations cannot negate the effect. Nor can the effect be duplicated by replacing infectious MCMV particles with UV-in-activated ones, indicating that mere surface contact between the 75 virus particles and the spleen cells during the adsorption period is insufficient. From the previous chapter, i t has been shown that macrophage-like cells can harbor the MCMV genome in a latent stage and permit a limited extent of v i r a l replication. Thus by infecting the macrophages in the spleen c e l l population, the v i r a l genome may redirect the cellular synthesizing act i v i t i e s and render them in -effective as mediators of T c e l l response to con A stimulation. The results of our c e l l mixing experiments indicated that this i s one plausible way to explain the suppression of con A response during MCMV infection. Peter Lipsky (Lipsky and Rosenthal, 1976), has suggested that the major function of macrophages towards regulating B c e l l DNA synthesis may be a negative one, acting to limit B c e l l proliferation. If so, then virus-induced release of B c e l l inhibitors early in infection may be a logical explanation for the suppression of the LPS response of infected spleen cel l s . The washing necessary to remove non-adherent cells i n our c e l l mixing experiments would have removed the inhibitors necessary to suppress B c e l l proliferation whereas the infected non-adherent ce l l s , having been 'turned o f f early in infection, would remain so during the rest of the experiment. Thus this remains an inter-esting hypothesis to work on in the future. 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